CN103063766B - Construction method of Chinese herbal medicine Naoshuantong preparation high performance liquid chromatography (HPLC) finger-print and application thereof - Google Patents
Construction method of Chinese herbal medicine Naoshuantong preparation high performance liquid chromatography (HPLC) finger-print and application thereof Download PDFInfo
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Abstract
The invention discloses a construction method of a Chinese herbal medicine Naoshuantong preparation high performance liquid chromatography (HPLC) finger-print. The construction method of the Chinese herbal medicine Naoshuantong preparation HPLC finger-print comprises a Chinese herbal medicine Naoshuantong preparation HPLC finger-print one and a Chinese herbal medicine Naoshuantong preparation HPLC finger-print two. A complete finger-print feature of the Chinese herbal medicine Naoshuantong is formed through the HPLC finger-print one and preparation HPLC finger-print two. The invention also discloses a HPLC finger-print in the Chinese herbal medicine Naoshuantong and the application in the extract semi-finished product detection. The construction method adopts different mobile phase systems and different detection wavelengths, the Chinese herbal medicine Naoshuantong HPLC finger-print one and preparation HPLC finger-print two are respectively established to detect the whole five flavors of medicinal ingredients so that the quality of the Chinese herbal medicine Naoshuantong preparation semi-manufactured product and product thereof can be monitored overall, and the construction method can guarantee the stable, uniform and controllable quality.
Description
Technical field
The present invention relates to Pharmaceutical Analysis technical field, particularly construction method and the application thereof of the logical preparation HPLC fingerprint pattern of a kind of brain bolt made from natural plant crude drugs raw material.
Background technology
International community is " safe, effective, controlled " to the requirement of medicine.Existing traditional Chinese medicine quality standard, is to determine the true and false with traditional proterties discriminating and micro-discriminating to the quality control of Chinese patent drug, and good and bad with physics and chemistry Identification Evaluation, minority flavour of a drug detect and control its 1 ~ 2 index components.From Chinese Traditional Medicine viewpoint, detect the whole curative effect that any active component all can not reflect that compound preparation embodies, tcm treatment according to syndrome differentiation is used is flavour of a drug but not certain chemical composition.Finger-print is the understanding based on centering medicinal substances group mass action, obtains spectrum or the chromatogram of chemical composition of Chinese materia medica by means of the technology such as wave spectrum and chromatogram, adopt " total peak " thus mode judge that similarity degree reaches control object.Can utilize finger-print to express the quality of finished product, realize the Quality Control to technological operation and crude drug; Also can utilize fingerprint trace to its source to find the problem in technological operation and realize the quality requirements to crude drug GAP.
The logical preparation of brain bolt is compound Chinese medicinal preparation, cattail pollen, the radix paeoniae rubrathe, root tuber of aromatic turmeric, rhizoma Gastrodiae, Radix Rhapontici seu Radix Echinopsis gomi herbs, consists of, and has effect promoting blood circulation and removing obstruction in channels, dispelling pathogenic wind and eliminating phlegm, the sick apoplex involving the channels and collaterals acute stage of ishemic stroke and the convalescence that for wind phlegm stasis blood-arthralgia resistance train of thought, cause.In the production run at the logical preparation of brain bolt, the radix paeoniae rubrathe, root tuber of aromatic turmeric 2 taste medicinal materials adopt alcohol extraction process, and cattail pollen, rhizoma Gastrodiae, Radix Rhapontici seu Radix Echinopsis 3 taste medicinal materials adopt aqueous extraction-alcohol precipitation technology, therefore the existing liposoluble constituent of composition has again water soluble ingredient in its finished product, complicated component, polarity differs greatly.But the quality standard imperfection of the logical preparation of brain bolt, lacks method of quality control comprehensively and effectively at present, is difficult to the quality of overall monitor finished product, the stability of overall monitor production technology.Existing quality standard " discriminating " adopts thin-layered chromatography to differentiate cattail pollen, rhizoma Gastrodiae, the radix paeoniae rubrathe, Radix Rhapontici seu Radix Echinopsis 4 taste medicinal materials in prescription, " assay " adopts the content of Paeoniflorin in the high effective liquid chromatography for measuring ministerial drug radix paeoniae rubrathe, its weak point is: this standard operation is loaded down with trivial details, root tuber of aromatic turmeric is not detected; It is few that single assay method provides information, not to the chemical composition in gomi herbs carry out comprehensively, the detection of system.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency of the prior art, by the research to the logical preparation HPLC fingerprint pattern construction method of brain bolt, provide method of quality control and the application thereof of the logical preparation of a kind of brain bolt effectively reliably.
Technical scheme of the present invention is as described below: the construction method of the logical preparation HPLC fingerprint pattern of brain bolt disclosed by the invention, comprise and build the logical preparation HPLC fingerprint pattern one of brain bolt and the logical preparation HPLC fingerprint pattern two of brain bolt, by HPLC finger-print one and HPLC finger-print two, jointly formed the Fingerprints of the logical preparation of complete brain bolt.
The construction method of the logical preparation HPLC fingerprint pattern one of described brain bolt comprises the following steps:
(1) preparation of need testing solution: precision takes the logical preparation 1.0g～10.0g of brain bolt, extracts with methyl alcohol, filters, gets filtrate appropriate, and evaporate to dryness, is made into the logical preparation need testing solution one of brain bolt with methyl alcohol, preferably with 30% methanol solution, prepare;
(2) adopt high-efficient liquid phase chromatogram technique analysis need testing solution and reference substance solution, obtain HPLC finger-print one, chromatographic condition is: sample size 5～30 μ l; Chromatographic column is ODS C
18, 3 μ m; Mobile phase is acetonitrile-tetrahydrofuran-phosphate aqueous solution; Gradient elution; 0～70 minute, mobile phase acetonitrile-tetrahydrofuran-phosphate aqueous solution faded to 20:10:70 by 2:0:98; 20 ° of C of column temperature; Detecting wavelength 0～53min is 254nm, after 53min, wavelength is switched to 275nm.
Described step (1) is more specifically: get this product content appropriate, mix, get about 2.5g, accurately weighed, to put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, with methyl alcohol, supply the weight of less loss, shake up, filter.(1) precision measures subsequent filtrate 20ml, and decompression and solvent recovery is to dry, and residue adds 30% methyl alcohol and dissolves and be transferred in 10ml measuring bottle, add 30% methyl alcohol and be diluted to scale, shake up, centrifugal, get supernatant, with 0.45 μ m miillpore filter, filter, as finished product need testing solution one.
The logical preparation HPLC fingerprint pattern one of described brain bolt utilizes similarity evaluation to detect 27 total chromatographic peaks, and the total chromatographic peak that belongs to cattail pollen has 5 peaks, is respectively No. 1 peak, No. 3 peaks, No. 12 peaks, No. 20 peaks, No. 22 peaks; The total chromatographic peak that belongs to the radix paeoniae rubrathe has 10 peaks, is respectively No. 5 peaks, No. 7 peaks, No. 11 peaks, No. 15 peaks, No. 16 peaks, No. 21 peaks, No. 23 peaks, No. 24 peaks, No. 25 peaks, No. 27 peaks; It is No. 6 peaks that the total chromatographic peak that belongs to rhizoma Gastrodiae has 1 peak; The total chromatographic peak that belongs to Radix Rhapontici seu Radix Echinopsis has 6 peaks, is respectively No. 10 peaks, No. 13 peaks, No. 14 peaks, No. 18 peaks, No. 19 peaks, No. 26 peaks 6; It is No. 9 peaks that the total chromatographic peak that belongs to root tuber of aromatic turmeric has a peak, and the composition chromatographic peak of decocting altogether that does not belong to any medicinal material has 4 peaks, is respectively No. 2 peaks, No. 4 peaks, No. 8 peaks, No. 17 peaks.
The construction method of the logical preparation HPLC fingerprint pattern one of described brain bolt is further comprising the steps of:
(1) preparation of reference substance solution: it is appropriate that precision takes typhaneoside, Isorhamnetin-3-O-neohespeidoside, Paeoniflorin, Gastrodin, β-ecdysterone, adding methyl alcohol makes to dissolve, make mixing reference substance solution, wherein the concentration of typhaneoside, Isorhamnetin-3-O-neohespeidoside, Paeoniflorin, Gastrodin, β-ecdysterone is respectively 0.01～3mg/ml;
(2) adopt high-efficient liquid phase chromatogram technique analysis for reference substance solution, obtain reference substance finger-print, chromatographic condition is: sample size 5～30 μ l; Chromatographic column is ODS C
18, 3 μ m; Mobile phase is acetonitrile-tetrahydrofuran-phosphate aqueous solution; Gradient elution; 0～70 minute, mobile phase acetonitrile-tetrahydrofuran-phosphate aqueous solution faded to 20:10:70 by 2:0:98; 20 ° of C of column temperature; Detecting wavelength 0～53min is 254nm, after 53min, wavelength is switched to 275nm;
(3) utilize DAD detecting device and RRLC/MS/MS technological means to carry out synergy and peak purity detection to the chromatographic peak of HPLC finger-print one and reference substance finger-print, more definite by UV absorption curve: No. 6 peaks are Gastrodin, No. 7 peaks are gallic acid, No. 11 peaks are oxypaeoniflorin, No. 13 peaks are Ajugasterone C, No. 15 peaks are Paeoniflorin, No. 18 peaks are β-ecdysterone, No. 20 peaks are typhaneoside, No. 21 peaks are galloyl Paeoniflorin or galloyl albiflorin or the isomeride of the two, No. 22 peaks are Isorhamnetin-3-O-neohespeidoside, No. 25 peaks are benzoxy Paeoniflorin.
The construction method of the logical preparation HPLC fingerprint pattern two of described brain bolt comprises the following steps:
(1) preparation of need testing solution: precision takes the logical preparation 1.0g～10.0g of brain bolt, extracts with methyl alcohol, filters, gets filtrate appropriate, and evaporate to dryness, suitably purifies, and with methyl alcohol, is made into the logical preparation need testing solution two of brain bolt, preferably with 30% methanol solution, prepares;
(2) adopt the above-mentioned need testing solution of high-efficient liquid phase chromatogram technique analysis, obtain the logical preparation HPLC fingerprint pattern two of brain bolt, chromatographic condition is: sample size 5～30 μ l; Chromatographic column is ODS C
18, 3 μ m; Mobile phase is acetonitrile-phosphate aqueous solution; Gradient elution; 0～5 minute, mobile phase acetonitrile-phosphate aqueous solution faded to 55:45 by 30:70, and 5～20 minutes mobile phase acetonitrile-phosphate aqueous solutions are 55:45; 25 ° of C of column temperature; Detect wavelength 390nm.
Described step (1) is more specifically: get the logical preparation of brain bolt, mix, get about 2.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures subsequent filtrate 20ml, decompression and solvent recovery is to dry, residue adds 50% methenyl choloride 25ml jolting and extracts, divide water intaking layer, with methenyl choloride 25ml jolting, extract again, merge methenyl choloride extract, reclaim methenyl choloride to dry, residue quantitatively adds methyl alcohol 2ml, make to dissolve completely, centrifugal, get supernatant, with 0.45 μ m miillpore filter, filter, as finished product need testing solution two.
The logical preparation HPLC fingerprint pattern two of described brain bolt utilizes similarity evaluation to detect 5 total chromatographic peaks, and the total chromatographic peak that belongs to root tuber of aromatic turmeric is No. 5 peaks.Utilize DAD detecting device and RRLC/MS/MS technological means to carry out peak ownership to chromatographic peak: No. 2 peaks are Demethoxycurcumin.
The invention also discloses and utilize the application in the logical preparation of brain bolt and medicinal extract semi-manufacture thereof detect of HPLC finger-print that the construction method of the logical preparation HPLC fingerprint pattern of above-mentioned brain bolt obtains.
Beneficial effect of the present invention is:
(1) the present invention has set up the fingerprint pattern technology standard of the logical preparation of brain bolt, by the feature that has that it's too late at total peak in finger-print, the quality of overall monitor semi-manufacture and finished product effectively, the stability of monitoring production technology, guarantees stable, the homogeneous, controlled of its quality.
(2) the present invention adopts different flow phase system and the different wavelength that detects, and sets up respectively the logical preparation finger one of brain bolt and finger-print two, jointly detects the whole gomi herbs compositions in compound.The finger-print of setting up can overall monitor raw medicinal material, the quality of semi-manufacture and finished product, by the comparison of chromatographic fingerprint feature similarity degree, evaluate good and bad, investigate stability and consistance, made up the deficiency of existing method of quality control.
(3) the present invention has improved the logical preparation finished product of brain bolt, half-finished quality monitoring standard, has effectively avoided product counterfeiting, guarantees normal production, the circulation order of this kind; On basis of the present invention, can carry out the correlation research of finger-print information and drug activity information, thereby in depth illustrate the intrinsic chemical composition of product and the correlativity of said preparation curative effect.
(4) method of the present invention also has good feasibility, stability and reappearance.
Accompanying drawing explanation
Fig. 1 is the logical preparation HPLC fingerprint pattern one of brain bolt of the present invention;
Fig. 2 is the logical preparation HPLC fingerprint pattern two of brain bolt of the present invention;
Fig. 3 mixes reference substance to lead to brain bolt the chromatogram that preparation agent HPLC finger-print one is corresponding, and in figure, peak numbers 6,15,18,20,22 is designated as Gastrodin, Paeoniflorin, β-ecdysterone, typhaneoside, Isorhamnetin-3-O-neohespeidoside successively;
Fig. 4 is the logical preparation of brain bolt, cattail pollen, lacks cattail pollen negative control chromatogram, and in figure, arrow indication is from left to right in the logical preparation HPLC fingerprint pattern one of brain bolt, to belong to the peak number of the chromatographic peak of cattail pollen;
Fig. 5 is the logical preparation of brain bolt, radix paeoniae rubrathe medicinal material, lacks radix paeoniae rubrathe negative control chromatogram, and in figure, arrow indication is from left to right in the logical preparation HPLC fingerprint pattern one of brain bolt, to belong to the peak number of the chromatographic peak of the radix paeoniae rubrathe;
Fig. 6 is the logical preparation of brain bolt, Rhizoma Gastrodiae, lacks rhizoma Gastrodiae negative control chromatogram, and in figure, arrow indication is in the logical preparation HPLC fingerprint pattern one of brain bolt, to belong to the peak number of the chromatographic peak of rhizoma Gastrodiae;
Fig. 7 is the logical preparation of brain bolt, Radix Rhapontici seu Radix Echinopsis medicinal material, gaps and omissions reed negative control chromatogram, and in figure, arrow indication is from left to right in the logical preparation HPLC fingerprint pattern one of brain bolt, to belong to the peak number of the chromatographic peak of Radix Rhapontici seu Radix Echinopsis;
Fig. 8 is the logical preparation of brain bolt, root tuber of aromatic turmeric medicinal material, lacks root tuber of aromatic turmeric negative control chromatogram, and in figure, arrow indication is in the logical preparation fingerprint HPLC collection of illustrative plates one of brain bolt, to belong to the peak number of the chromatographic peak of root tuber of aromatic turmeric;
Fig. 9 is the logical preparation of brain bolt, root tuber of aromatic turmeric medicinal material, lacks root tuber of aromatic turmeric negative control chromatogram, and in figure, arrow indication is in the logical preparation HPLC fingerprint pattern two of brain bolt, to belong to the peak number of the chromatographic peak of root tuber of aromatic turmeric.
Embodiment
Below in conjunction with specific embodiment, describe the present invention.
1, instrument and reagent
1.1 instruments: the Dionex Ultimate 3000DGLC of U.S. Dai An company high performance liquid chromatograph (the two ternary pumps of DGP-3600SD, SRD-3600 degasser, WPS-3000SL automatic sampler, TCC3000-RS column oven, PDA-3000 detecting device); Chromatographic column: Ultimate AQ-C
18(4.6 * 150mm, 3 μ m).
1.2 reagents: 10 batches of finished products and corresponding semi-manufacture, provide by GuangDong HuaNan Pharmacy Group Co., Ltd.In experiment, liquid chromatography agents useful for same acetonitrile, tetrahydrofuran are chromatographically pure, and water is ultrapure water, and it is pure that all the other agents useful for same are analysis.
1.3 reference substances:
2, method and result
The construction method of the logical preparation HPLC fingerprint pattern one of 2.1 brain bolts
2.1.1 the preparation of solution
The preparation of the logical preparation need testing solution of brain bolt: get Naoshuantong capsule content appropriate, mix, get 2.5g, put in tool plug conical flask, accurately weighed, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures subsequent filtrate 20ml, decompression and solvent recovery is to dry, residue adds 30% methyl alcohol and dissolves and be transferred in 10ml measuring bottle, add 30% methyl alcohol and be diluted to scale, shake up, centrifugal, get supernatant, with 0.45 μ m miillpore filter, filter, as need testing solution one, Naoshuantong capsule content content is 0.1g/ml,
Mix the preparation of reference substance solution: get typhaneoside, Isorhamnetin-3-O-neohespeidoside, Paeoniflorin, Gastrodin, β-ecdysterone reference substance appropriate, accurately weighed, add 30% methyl alcohol and make every 1ml containing the mixed solution of typhaneoside, Isorhamnetin-3-O-neohespeidoside, Paeoniflorin, Gastrodin, each 0.1mg of β-ecdysterone;
The preparation of medicinal material need testing solution: get above-mentioned ingredients meal, sampling amount is respectively cattail pollen 5.56g, radix paeoniae rubrathe 3.97g, root tuber of aromatic turmeric 3.19g, rhizoma Gastrodiae 1.59g, Radix Rhapontici seu Radix Echinopsis 2.38g, prepares respectively single medicinal material need testing solution.After extracting by the method for making of production technology respectively, solution decompression evaporate to dryness, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures subsequent filtrate 20ml, and decompression and solvent recovery is to dry, residue adds 30% methyl alcohol and dissolves and be transferred in 10ml measuring bottle, add 30% methyl alcohol and be diluted to scale, shake up, centrifugal, get supernatant, with 0.45 μ m miillpore filter, filter, as medicinal material need testing solution, standby.
The preparation of negative need testing solution: get respectively and lack cattail pollen negative sample 1.67g, lack radix paeoniae rubrathe negative sample 1.91g, lack root tuber of aromatic turmeric negative sample 2.02g, lack rhizoma Gastrodiae negative sample 2.26g, gaps and omissions reed negative sample 2.14g, by " preparation of the logical preparation need testing solution of a brain bolt " lower method operation, obtains.
2.1.2 efficient liquid phase chromatographic analysis: the accurate logical preparation need testing solution 10 μ l of brain bolt, the sample introduction drawn; Chromatographic condition: chromatographic column is Ultimate AQ-C
18(4.6 * 150mm, 3 μ m); Mobile phase is acetonitrile (A)-tetrahydrofuran (B)-0.05% phosphoric acid solution (C), adopts following gradient elution mode:
Detecting wavelength: 0～53min is 254nm, after 53min, wavelength is switched to 275nm; Flow velocity: 1.1ml/min; Column temperature: 20 ° of C; Obtain the logical preparation HPLC fingerprint pattern one of brain bolt, as shown in Figure 1.
2.1.3 contrast color spectrum analysis
To the logical preparation of brain bolt, mix reference substance, and single medicinal material and the negative need testing solution medicine that lacks this medicinal material carry out respectively the detection of contrast color spectrogram, as shown in Fig. 3-Fig. 8.
Wherein Fig. 3 mixes reference substance to lead to brain bolt the chromatogram that preparation finger one is corresponding, and in figure, peak numbers 6,15,18,20,22 is designated as Gastrodin, Paeoniflorin, β-ecdysterone, typhaneoside, Isorhamnetin-3-O-neohespeidoside successively.
Fig. 4 is the logical preparation of brain bolt, cattail pollen, lacks cattail pollen negative control chromatogram, and in figure, arrow indication is from left to right in the logical preparation finger one of brain bolt, to belong to the peak number of the chromatographic peak of cattail pollen.
Fig. 5 is the logical preparation of brain bolt, radix paeoniae rubrathe medicinal material, lacks radix paeoniae rubrathe negative control chromatogram, and in figure, arrow indication is from left to right in the logical preparation finger one of brain bolt, to belong to the peak number of the chromatographic peak of the radix paeoniae rubrathe.
Fig. 6 is the logical preparation of brain bolt, Rhizoma Gastrodiae, lacks rhizoma Gastrodiae negative control chromatogram, and in figure, arrow indication is in the logical preparation finger one of brain bolt, to belong to the peak number of the chromatographic peak of rhizoma Gastrodiae.
Fig. 7 is the logical preparation of brain bolt, Radix Rhapontici seu Radix Echinopsis medicinal material, gaps and omissions reed negative control chromatogram, and in figure, arrow indication is from left to right in the logical preparation finger one of brain bolt, to belong to the peak number of the chromatographic peak of Radix Rhapontici seu Radix Echinopsis.
Fig. 8 is the logical preparation of brain bolt, root tuber of aromatic turmeric medicinal material, lacks root tuber of aromatic turmeric negative control chromatogram, and in figure, arrow indication is in the logical preparation finger one of brain bolt, to belong to the peak number of the chromatographic peak of root tuber of aromatic turmeric.
2.1.4 total peak is determined: the efficient liquid-phase chromatograph finger print atlas of the logical preparation of 10 batches of brain bolts obtained above is compared through < < similarity evaluation > > (2009 editions), determine 27 common characteristic peaks and obtain common pattern (with reference to finger-print).The HPLC finger-print one of gained detect with write out a prescription in cattail pollen, the radix paeoniae rubrathe, rhizoma Gastrodiae, Radix Rhapontici seu Radix Echinopsis, root tuber of aromatic turmeric medicinal material is as follows with reference to the identical chromatographic peak of finger-print: the total peak that belongs to cattail pollen is No. 1 peak, No. 3 peaks, No. 12 peaks, No. 20 peaks, Wu Ge peak, No. 22 peaks, the total peak that belongs to the radix paeoniae rubrathe is No. 5 peaks, No. 7 peaks, No. 11 peaks, No. 15 peaks, No. 16 peaks, No. 21 peaks, No. 23 peaks, No. 24 peaks, No. 25 peaks, Shi Ge peak, No. 27 peaks, the total peak that belongs to rhizoma Gastrodiae is Yi Ge peak, No. 6 peaks, the total peak that belongs to Radix Rhapontici seu Radix Echinopsis is No. 10 peaks, No. 13 peaks, No. 14 peaks, No. 18 peaks, No. 19 peaks, Liu Ge peak, No. 26 peaks, the total peak that belongs to root tuber of aromatic turmeric is Yi Ge peak, No. 9 peaks, the composition chromatographic peak of decocting altogether that does not belong to any medicinal material is No. 2 peaks, No. 4 peaks, No. 8 peaks, Si Ge peak, No. 17 peaks.Utilize DAD detecting device and RRLC/MS/MS technological means to carry out synergy and peak purity detection to main peaks, by the comparison of UV absorption curve, in conjunction with mass spectrographic molecular ion peak, cleaved fragment information is identified: No. 6 peaks are Gastrodin, No. 7 peaks are gallic acid, No. 11 peaks are oxypaeoniflorin, No. 13 peaks are Ajugasterone C, No. 15 peaks are Paeoniflorin, No. 18 peaks are β-ecdysterone, No. 20 peaks are typhaneoside, No. 21 peaks are galloyl Paeoniflorin/galloyl albiflorin or its isomeride, No. 22 peaks are Isorhamnetin-3-O-neohespeidoside, No. 25 peaks are benzoxy Paeoniflorin.Above chromatographic peak has formed the fingerprint characteristic of the logical preparation HPLC fingerprint pattern one of brain bolt.
No. 15 peak Paeoniflorins of take are object of reference peak, and the relative retention value at Naoshuantong capsule HPLC finger-print one total peak is listed as follows:
2.1.5 precision test: get the logical preparation need testing solution one of same brain bolt, continuous sample introduction 6 times, detect finger-print, adopt < < similarity evaluation > > (2009 editions) to evaluate, similarity is all greater than 0.99, shows that the precision of instrument is good.
Similarity result is as follows:
| ? | |
|
Precision 3 | |
Precision 5 | |
|
| Precision | |||||||
| 1 | 1.000 | 0.997 | 0.988 | 0.988 | 0.990 | 0.993 | 0.996 |
| |
0.997 | 1.000 | 0.992 | 0.990 | 0.990 | 0.997 | 0.997 |
| Precision 3 | 0.988 | 0.992 | 1.000 | 0.998 | 0.996 | 0.992 | 0.997 |
| |
0.988 | 0.990 | 0.998 | 1.000 | 0.997 | 0.990 | 0.997 |
| Precision 5 | 0.990 | 0.990 | 0.996 | 0.997 | 1.000 | 0.991 | 0.997 |
| |
0.993 | 0.997 | 0.992 | 0.990 | 0.991 | 1.000 | 0.997 |
| Reference fingerprint | 0.996 | 0.997 | 0.997 | 0.997 | 0.997 | 0.997 | 1.000 |
2.1.6 study on the stability: get the logical preparation need testing solution one of same brain bolt, placing 0,2,4,8,10,24,48 hour sample introduction respectively, detect finger-print, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.99, shows that need testing solution places in 48 hours stable.
Similarity result is as follows:
| ? | S1(0h) | S2(2h) | S3(4h) | S4(8h) | S5(12h) | S6(24h) | S7(48h) | Reference fingerprint |
| S1(0h) | 1.000 | 0.997 | 0.988 | 0.990 | 0.988 | 0.990 | 0.987 | 0.994 |
| S2(2h) | 0.997 | 1.000 | 0.992 | 0.990 | 0.991 | 0.991 | 0.990 | 0.996 |
| S3(4h) | 0.988 | 0.992 | 1.000 | 0.996 | 0.997 | 0.995 | 0.997 | 0.998 |
| S4(8h) | 0.990 | 0.990 | 0.996 | 1.000 | 0.997 | 0.998 | 0.996 | 0.998 |
| S5(12h) | 0.988 | 0.991 | 0.997 | 0.997 | 1.000 | 0.997 | 0.998 | 0.998 |
| S6(24h) | 0.990 | 0.991 | 0.995 | 0.998 | 0.997 | 1.000 | 0.995 | 0.998 |
| S7(48h) | 0.987 | 0.990 | 0.997 | 0.996 | 0.998 | 0.995 | 1.000 | 0.998 |
| Reference fingerprint | 0.994 | 0.996 | 0.998 | 0.998 | 0.998 | 0.998 | 0.998 | 1.000 |
2.17 replica tests: get with 6 parts, the logical preparation of a collection of brain bolt, press method operation under need testing solution preparation, difference sample introduction, detect finger-print, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.99, shows that method is reproducible.
Similarity result is as follows:
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Reference fingerprint |
| S1 | 1.000 | 0.997 | 0.992 | 0.996 | 0.996 | 0.995 | 0.998 |
| S2 | 0.997 | 1.000 | 0.998 | 0.998 | 0.996 | 0.996 | 0.999 |
| S3 | 0.992 | 0.998 | 1.000 | 0.996 | 0.993 | 0.993 | 0.997 |
| S4 | 0.996 | 0.998 | 0.996 | 1.000 | 0.999 | 0.994 | 0.999 |
| S5 | 0.996 | 0.996 | 0.993 | 0.999 | 1.000 | 0.994 | 0.998 |
| S6 | 0.995 | 0.996 | 0.993 | 0.994 | 0.994 | 1.000 | 0.997 |
| Reference fingerprint | 0.998 | 0.999 | 0.997 | 0.999 | 0.998 | 0.997 | 1.000 |
2.1.8 precision in the middle of: precision takes the logical preparation of brain bolt of same lot number, respectively under the variable condition such as same date, different analyst, different instruments not, measure in accordance with the law, detect finger-print, adopt " similarity evaluation " (2009 editions) to evaluate.In the middle of showing the method, precision is good, and logical preparation HPLC fingerprint pattern one method of quality control of brain bolt is feasible.
(1) different analysis times: the logical preparation of brain bolt of getting same lot number, respectively at same date not by " preparation of need testing solution " lower method operation, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.99.
Similarity result is as follows:
| ? | |
|
Reference |
| Analysis time | |||
| 1 | 1.000 | 0.970 | 0.992 |
| |
0.970 | 1.000 | 0.993 |
| Reference fingerprint | 0.992 | 0.993 | 1.000 |
(2) different analysts: the logical preparation of brain bolt of getting same lot number, different personnel are respectively by " preparation of a need testing solution " lower method operation, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.99.
Similarity result is as follows:
| ? | |
|
|
| Analyst | |||
| 1 | 1.000 | 0.997 | 0.999 |
| |
0.997 | 1.000 | 0.999 |
| Reference fingerprint | 0.999 | 0.999 | 1.000 |
The construction method of the logical preparation HPLC fingerprint pattern two of 2.2 brain bolts
2.2.1 the preparation of solution
The preparation of the logical preparation need testing solution of brain bolt: get this product content appropriate, porphyrize, get about 2.5g, put in tool plug conical flask, accurately weighed, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures subsequent filtrate 20ml, decompression and solvent recovery is to dry, residue adds 50% methenyl choloride 25ml jolting and extracts, divide water intaking layer, with methenyl choloride 25ml jolting, extract again, merge methenyl choloride extract, reclaim methenyl choloride to dry, residue quantitatively adds methyl alcohol 2ml, make to dissolve completely, centrifugal, get supernatant, with 0.45 μ m miillpore filter, filter, as finished product need testing solution two (0.5g/ml),
The preparation of medicinal material need testing solution: get root tuber of aromatic turmeric medicinal material coarse powder 3.19g, after extracting by the method for making of production technology, solution decompression evaporate to dryness, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures subsequent filtrate 20ml, decompression and solvent recovery is to dry, residue adds 50% methenyl choloride 25ml jolting and extracts, divide water intaking layer, with methenyl choloride 25ml jolting, extract again, merge methenyl choloride extract, reclaim methenyl choloride to dry, residue quantitatively adds methyl alcohol 2ml, make to dissolve completely, centrifugal, get supernatant, with 0.45 μ m miillpore filter, filter, as medicinal material need testing solution, standby.
The preparation of negative need testing solution: get and lack root tuber of aromatic turmeric negative sample 2.02g, by " preparation of the logical preparation need testing solution of a brain bolt " lower method operation, obtain.
2.2.2 efficient liquid phase chromatographic analysis: accurate need testing solution 10 μ l, the sample introduction drawn; Chromatographic condition: chromatographic column is Ultimate AQ-C
18(4.6 * 150mm, 3 μ m); Mobile phase is acetonitrile (A)-0.05% phosphate aqueous solution (B), adopts following gradient elution mode:
Detect wavelength: 390nm; Flow velocity: 1.0ml/min; Column temperature: 25 ° of C; Obtain the logical preparation HPLC fingerprint pattern two of brain bolt, as shown in Figure 2.
2.2.3 contrast chromatogram detects
Fig. 9 is the logical preparation of brain bolt, root tuber of aromatic turmeric medicinal material, lacks root tuber of aromatic turmeric negative control chromatogram, and in figure, arrow indication is in the logical preparation finger two of brain bolt, to belong to the peak number of the chromatographic peak of root tuber of aromatic turmeric.
2.2.4 total peak is determined: the logical preparation HPLC fingerprint pattern two of 10 batches of brain bolts obtained above is compared through < < similarity evaluation > > (2009 editions), determine 5 common characteristic peaks and obtain common pattern (with reference to finger-print).The chromatogram of gained should detect with write out a prescription in root tuber of aromatic turmeric medicinal material as follows with reference to the identical chromatographic peak of finger-print: the total peak that belongs to root tuber of aromatic turmeric is Yi Ge peak, No. 5 peaks; Utilize DAD detecting device and RRLC/MS/MS technological means to carry out peak ownership to chromatographic peak: No. 2 peaks are Demethoxycurcumin.Above chromatographic peak has formed the fingerprint characteristic of the logical preparation HPLC fingerprint pattern one of brain bolt.
No. 2 peak Demethoxycurcumins of take are object of reference peak, and the relative retention value at Naoshuantong capsule finger-print two total peaks is listed as follows:
| Peak number | Relative retention value | Ownership medicinal material | Chromatographic |
| 1 | 0.3 | - | - |
| 2 | 1.0 | Root tuber of aromatic turmeric | Demethoxycurcumin |
| 3 | 1.3 | - | - |
| 4 | 1.6 | - | - |
| 5 | 1.8 | Root tuber of aromatic turmeric | - |
2.2.5 precision test: get the logical preparation need testing solution two of same brain bolt, continuous sample introduction 6 times, detect finger-print, adopt < < similarity evaluation > > (2009 editions) to evaluate, each similarity detecting under wavelength is all greater than 0.999, shows that the precision of instrument is good.
Similarity result is as follows:
| ? | |
|
Precision 3 | |
Precision 5 | |
|
| Precision | |||||||
| 1 | 1.000 | 0.998 | 0.998 | 0.998 | 0.998 | 0.998 | 0.999 |
| |
0.998 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Precision 3 | 0.998 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| |
0.998 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Precision 5 | 0.998 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| |
0.998 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Reference fingerprint | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
2.2.6 study on the stability: get the logical preparation need testing solution two of same brain bolt, respectively at 0,2,4,8,10,24,48 hour sample introduction, detect finger-print, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.999, shows that need testing solution places in 48 hours stable.Similarity result is as follows:
| ? | S1(0h) | S2(2h) | S3(4h) | S4(8h) | S5(12h) | S6(24h) | S7(48h) | Reference fingerprint |
| S1(0h) | 1.000 | 0.998 | 0.998 | 0.997 | 0.997 | 0.999 | 0.999 | 0.999 |
| S2(2h) | 0.998 | 1.000 | 1.000 | 1.000 | 0.999 | 0.998 | 0.999 | 1.000 |
| S3(4h) | 0.998 | 1.000 | 1.000 | 1.000 | 0.999 | 0.997 | 0.999 | 1.000 |
| S4(8h) | 0.997 | 1.000 | 1.000 | 1.000 | 1.000 | 0.997 | 0.999 | 1.000 |
| S5(12h) | 0.997 | 0.999 | 0.999 | 1.000 | 1.000 | 0.997 | 0.998 | 0.999 |
| S6(24h) | 0.999 | 0.998 | 0.997 | 0.997 | 0.997 | 1.000 | 0.999 | 0.999 |
| S7(48h) | 0.999 | 0.999 | 0.999 | 0.999 | 0.998 | 0.999 | 1.000 | 1.000 |
| Reference fingerprint | 0.999 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 |
2.2.7 replica test: get with 6 parts, the logical preparation of a collection of brain bolt, press method operation under need testing solution preparation, difference sample introduction, detect finger-print, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.92, shows that method is reproducible.
Similarity result is as follows:
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Reference fingerprint |
| S1 | 1.000 | 0.993 | 0.997 | 0.988 | 0.942 | 0.988 | 0.998 |
| S2 | 0.993 | 1.000 | 0.988 | 0.971 | 0.969 | 0.971 | 0.990 |
| S3 | 0.997 | 0.988 | 1.000 | 0.996 | 0.925 | 0.995 | 1.000 |
| S4 | 0.988 | 0.971 | 0.996 | 1.000 | 0.887 | 1.000 | 0.995 |
| S5 | 0.942 | 0.969 | 0.925 | 0.887 | 1.000 | 0.887 | 0.928 |
| S6 | 0.988 | 0.971 | 0.995 | 1.000 | 0.887 | 1.000 | 0.995 |
| Reference fingerprint | 0.998 | 0.990 | 1.000 | 0.995 | 0.928 | 0.995 | 1.000 |
2.2.8 precision in the middle of: precision takes the logical preparation of brain bolt of same lot number, respectively under the variable condition such as same date, different analyst, different instruments not, measure in accordance with the law, detect finger-print, adopt " similarity evaluation " (2009 editions) to evaluate.In the middle of showing the method, precision is good, and logical preparation HPLC fingerprint pattern two method of quality control of brain bolt are feasible.
(1) different analysis times: the logical preparation of brain bolt of getting same lot number, respectively at same date not by " preparation of need testing solution " lower method operation, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.99.
Similarity result is as follows:
| ? | |
|
Reference |
| Analysis time | |||
| 1 | 1.000 | 0.988 | 0.995 |
| |
0.988 | 1.000 | 0.999 |
| Reference fingerprint | 0.995 | 0.999 | 1.000 |
(2) different analysts: the logical preparation of brain bolt of getting same lot number, different personnel are respectively by " preparation of a need testing solution " lower method operation, adopt " similarity evaluation " (2009 editions) to evaluate, similarity is all greater than 0.99.
Similarity result is as follows:
| ? | |
|
|
| Analyst | |||
| 1 | 1.000 | 0.991 | 0.999 |
| |
0.991 | 1.000 | 0.996 |
| Reference fingerprint | 0.999 | 0.996 | 1.000 |
The present invention adopts different flow phase system and the different wavelength that detects, and sets up respectively the logical preparation finger one of brain bolt and finger-print two, jointly detects the whole gomi herbs compositions in compound.
The present invention not only can utilize the construction method of the logical preparation HPLC fingerprint pattern of above-mentioned brain bolt to be applied in the quality testing of the logical preparation of brain bolt, can also be applied in the logical preparation medicinal extract semi-manufacture detection of brain bolt, the quality of overall monitor semi-manufacture and finished product effectively, monitor the stability of production technology, guarantee stable, the homogeneous, controlled of its quality.
In sum; although the present invention have been described in detail the present invention by embodiment; but persons skilled in the art should be understood that; above-described embodiment is only the description to the preferred embodiments of the present invention; but not limiting the scope of the invention; persons skilled in the art are in the disclosed technical scope of the present invention, and the variation that can expect easily, all within protection scope of the present invention.
Claims (4)
1. the construction method of the logical preparation HPLC fingerprint pattern of a brain bolt, it is characterized in that: comprise and build the logical preparation HPLC fingerprint pattern one of brain bolt and the logical preparation HPLC fingerprint pattern two of brain bolt, the Fingerprints that has jointly formed the logical preparation of complete brain bolt by HPLC finger-print one and HPLC finger-print two, the construction method of the logical preparation HPLC fingerprint pattern one of described brain bolt comprises the following steps:
(1) preparation of need testing solution: precision takes the logical preparation 1.0g～10.0g of brain bolt, extracts with methyl alcohol, filters, gets filtrate appropriate, and evaporate to dryness, is made into the logical preparation need testing solution one of brain bolt with methyl alcohol;
(2) adopt high-efficient liquid phase chromatogram technique analysis need testing solution, obtain HPLC finger-print one, chromatographic condition is: sample size 5～30 μ l; Chromatographic column is ODS C
18, 3 μ m; Mobile phase is acetonitrile-tetrahydrofuran-phosphate aqueous solution; Gradient elution; 0～70 minute, mobile phase acetonitrile-tetrahydrofuran-phosphate aqueous solution faded to 20:10:70 by 2:0:98; 20 ℃ of column temperatures; Detecting wavelength 0～53min is 254nm, after 53min, wavelength is switched to 275nm;
The construction method of the logical preparation HPLC fingerprint pattern one of described brain bolt is further comprising the steps of:
(1) preparation of reference substance solution: it is appropriate that precision takes typhaneoside, Isorhamnetin-3-O-neohespeidoside, Paeoniflorin, Gastrodin, β-ecdysterone, adding methyl alcohol makes to dissolve, make mixing reference substance solution, wherein the concentration of typhaneoside, Isorhamnetin-3-O-neohespeidoside, Paeoniflorin, Gastrodin, β-ecdysterone is respectively 0.01～3mg/ml;
(2) adopt high-efficient liquid phase chromatogram technique analysis reference substance solution, obtain reference substance finger-print, chromatographic condition is: sample size 5～30 μ l; Chromatographic column is ODS C
18, 3 μ m; Mobile phase is acetonitrile-tetrahydrofuran-phosphate aqueous solution; Gradient elution; 0～70 minute, mobile phase acetonitrile-tetrahydrofuran-phosphate aqueous solution faded to 20:10:70 by 2:0:98; 20 ℃ of column temperatures; Detecting wavelength 0～53min is 254nm, after 53min, wavelength is switched to 275nm;
(3) utilize DAD detecting device and RRLC/MS/MS technological means to carry out synergy and peak purity detection to the chromatographic peak of HPLC finger-print one and reference substance finger-print, more definite by UV absorption curve: No. 6 peaks are Gastrodin, No. 7 peaks are gallic acid, No. 11 peaks are oxypaeoniflorin, No. 13 peaks are Ajugasterone C, No. 15 peaks are Paeoniflorin, No. 18 peaks are β-ecdysterone, No. 20 peaks are typhaneoside, No. 21 peaks are galloyl Paeoniflorin or galloyl albiflorin or the isomeride of the two, No. 22 peaks are Isorhamnetin-3-O-neohespeidoside, No. 25 peaks are benzoxy Paeoniflorin,
The construction method of the logical preparation HPLC fingerprint pattern two of described brain bolt comprises the following steps:
(1) preparation of need testing solution: precision takes the logical preparation 1.0g～10.0g of brain bolt, extracts with methyl alcohol, filters, gets filtrate appropriate, and evaporate to dryness, suitably purifies, and with methyl alcohol, is made into the logical preparation need testing solution two of brain bolt;
(2) adopt the above-mentioned need testing solution of high-efficient liquid phase chromatogram technique analysis, obtain the logical preparation HPLC fingerprint pattern two of brain bolt, chromatographic condition is: sample size 5～30 μ l; Chromatographic column is ODS C
18, 3 μ m; Mobile phase is acetonitrile-phosphate aqueous solution; Gradient elution; 0～5 minute, mobile phase acetonitrile-phosphate aqueous solution faded to 55:45 by 30:70, and 5～20 minutes mobile phase acetonitrile-phosphate aqueous solutions are 55:45; 25 ℃ of column temperatures; Detect wavelength 390nm;
The logical preparation HPLC fingerprint pattern two of described brain bolt utilizes similarity evaluation to detect 5 total chromatographic peaks, the total chromatographic peak that belongs to root tuber of aromatic turmeric is No. 5 peaks, utilizes DAD detecting device and RRLC/MS/MS technological means to carry out peak ownership to chromatographic peak: No. 2 peaks are Demethoxycurcumin.
2. brain bolt as claimed in claim 1 leads to the construction method of preparation HPLC fingerprint pattern, it is characterized in that: the logical preparation HPLC fingerprint pattern one of described brain bolt utilizes similarity evaluation to detect 27 total chromatographic peaks, the total chromatographic peak that belongs to cattail pollen has 5 peaks, is respectively No. 1 peak, No. 3 peaks, No. 12 peaks, No. 20 peaks, No. 22 peaks; The total chromatographic peak that belongs to the radix paeoniae rubrathe has 10 peaks, is respectively No. 5 peaks, No. 7 peaks, No. 11 peaks, No. 15 peaks, No. 16 peaks, No. 21 peaks, No. 23 peaks, No. 24 peaks, No. 25 peaks, No. 27 peaks; It is No. 6 peaks that the total chromatographic peak that belongs to rhizoma Gastrodiae has 1 peak; The total chromatographic peak that belongs to Radix Rhapontici seu Radix Echinopsis has 6 peaks, is respectively No. 10 peaks, No. 13 peaks, No. 14 peaks, No. 18 peaks, No. 19 peaks, No. 26 peaks; It is No. 9 peaks that the total chromatographic peak that belongs to root tuber of aromatic turmeric has 1 peak, and the composition chromatographic peak of decocting altogether that does not belong to any medicinal material has 4 peaks, is respectively No. 2 peaks, No. 4 peaks, No. 8 peaks, No. 17 peaks.
3. the construction method of the logical preparation HPLC fingerprint pattern of brain bolt as claimed in claim 1, is characterized in that: the methanol concentration used of preparation need testing solution and reference substance solution is 30%.
4. utilize the application in the logical preparation of brain bolt and medicinal extract semi-manufacture thereof detect of HPLC finger-print that the construction method of the logical preparation HPLC fingerprint pattern of brain bolt described in claim 1 obtains.
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